The overall goal of this proposal is to develop a novel imaging system based on the nonlinear optical process called higher harmonic generation. In this process two (or three) photons combine to form a single photon with twice (or three times) the frequency of the original photons that formed it. This phenomenon results from the interaction between photons from a high intensity source and the nonlinear optical properties of the object. Measurements of this "frequency-doubled and tripled" light can provide detailed information about structural and spectral properties of the object from which it emanates. In the present application we propose to use a femtosecond-pulsed laser to access an immense biological database, the extracellular matrix. There are compelling reasons for this choice. First, the matrix is far from an inert infrastructure. Rather, there is a constant reciprocal flow of information between matrix and resident cell population that reflects current physiological status. Second, collagen (the predominant component of the matrix) is, unlike most proteins, a molecule of infinite variety. With more than seventeen genetically distinct forms and an extensive menu of post-translational modifications, collagen can be thought of as a kind of structural chameleon, reflecting changes in its environment biochemically rather than chromatically. Many pathological conditions are associated with well-characterized structural changes in collagen. For example, melanoma is associated with loss of fibrillar organization of dermal collagen; diabetic complications with increased nonenzymatic glycation of collagen, and hypertrophic scarring with increased lysine hydroxylation. Our preliminary data document that these changes can be detected by higher harmonic analysis. We have assembled a uniquely strong research team to pursue these promising findings, with the goal of developing a clinically useful diagnostic tool with wide range of potential applications. To this end we propose to accomplish the following Specific Aims: 1. Design a prototype nonlinear confocal imaging system with the necessary compactness, flexibility, and sensitivity for clinical use. 2. Conduct a systematic survey of collagen preparations (purified and in native tissue) that will allow us to identify optical signatures of those structural features indicative of pathophysiological conditions. 3. Develop and refine mathematical models that will allow predictive inference about matrix structure from optical data. 4. Construct portable confocal system and begin clinical data collection.